1. Field of the Invention
The present invention relates to a method for preparing lysophosphatidylethanolamine from phospholipid mixture with a high purity. More specifically, the present invention relates to a method for purifying lysophosphatidylethanolamine with a high purity by treating phospholipid mixture with enzyme then applying solvent fractionation, without column purification.
2. Description of the Prior Art
Lysophosphatidylethanolamine exists naturally in animal cells or plant cells, and is plentiful in egg yolk or brain cell. The lysophosphatidylethanolamine is derived from phosphatidylethanolamine, which is a kind of phospholipids and detected in cell membrane. Phosphatidyletlanolamine, rich in egg yolk or soybean lecithin, is a kind of phospholipid containing two fatty acids in its molecule. In organisms, when Phospholipase A2, a kind of phospholipid hydrolase, acts on phosphatidylethanolamine, one fatty acid of which positioned in the sn-2 site is eliminated to produce lysophosphatidylethanolamine.
Lysophosphatidylethanolamine is known as playing an important role in the ripeness and senescence of fruit. It is well known that treating tomato plant with lysophosphatidylethanolamine suppresses senescence of their leaves and fruit, and that treating harvested tomato with lysophosphatidylethanolamine extends their storage term (U.S. Pat. Nos. 5,110,341, 5,126,155). It is also known that treating an apple with lysophosphatidylethanolamine promotes the formation of anthocyanin in the skin of the apple, and suppresses the loss of firmness when storing. Further, it is known that the above-described effects relate to the function of the lysophosphatidylethanolamine such as reducing the respiration rate of fruit(for example, apple, cranberry, tomato, etc.) and promoting or suppressing the formation of ethylene gas (Farag. K. M. and J. P. Palta, xe2x80x9cStimulation of Ethylene Production by Erea, Thidiazoron, and Lysophosphatidylethanolamine and Possible sites of this stimulationxe2x80x9d Annual meeting of the American Society of Plant Physiologists, April 1989).
The method of treating with lysophosphatidylethanolamine solution, which is controlled to have an appropriate concentration, has been used to prolong the life of the cut flowers (Hort Science 32(5): 888-890, 1997). Recently, silver thiosulfate solution, generally containing sugar, has been used for the purpose of suppressing the senescence of flowers by the method of treating the harvested (cut) flowers with the solution for 20 hours or more. The above solution, however, has the drawback that silver ion contained therein causes environmental pollutions. Because lysophosphatidylethanolamine purified from the nature, like the silver thiosulfate solution, has a characteristic of increasing the shelf life of the cut flower in a vase, lysophosphatidylethanolamine has been a target of wide investigation in this field.
Until now, the industrial method for producing lysophosphatidylethanolamine with high purity has not been developed. Just a small scale of isolation and purification of the lysophosphatidylethanolamine, using silica gel column chromatography, was performed in the laboratories. A little amount of lysophosphatidylethanolamine, just as for a reagent, is sold by Avanti Polar Lipids, Inc. or by Sigma Chemical Co., in a high price. Furthermore, when lysophosphatidylethanolamine is prepared by column chromatography, because lysophosphatidylcholin and lysophosphatidylethanolamine have similar patterns of migration, it is very difficult to isolate lysophosphatidylethanolamine from the two. When performing column chromatography, using a low-toxic organic solvent such as hexane or ethanol as a single solvent, because the solubility of the lysophosphatidylethanolamine to the solvent is very low, it is very difficult to purify lysophosphatidylethanolamine. Meanwhile, when chromatography is performed with using the solvent which is known as having a high toxicity (for example, chloroform, benzene or methanol), the result of the purification is good but the yield is low and the purification cost is high.
Thus, an object of the present invention is to provide a method for preparing lysophosphatidylethanolamine, with a high purity.
To accomplish the above object the present invention provides a method for preparing lysophosphatidylethanolamine comprising the steps of; treating phospholipid mixture comprising phosphatidylethanolamine with phospholipase to convert said phosphatidylethanolamine into the lysophosphatidylethanolamine; treating above-obtained lysophospholipid mixture comprising the lysophosphatidylethanolamine with a specific solvent mixture to extract and crystallize the lysophosphatidylethanolamine selectively; and recovering the lysophosphatidylethanolamine.
The present invention also provides a method further comprising the step of treating phosphatidylcholine, which is plentiful in the phospholipid, with phospholipase D, a kind of phospholipid hydrolyzing enzyme, to magnify the content of phosphatidylethanolamine in phospholipid mixture.
These and other features of the present invention will be well understood by one skilled in the art from the following detailed descriptions.